From engineering to evolution of synthetic cells with RNA origami

Introduction

Can we construct a cell from non-living matter? In search for answers, bottom-up synthetic biology has successfully encapsulated functional sets of biomolecules inside lipid vesicles, yet a “living” synthetic cell remains unattained. ENSYNC aims for a prototype of a synthetic cell that encompasses a fundamental characteristic of life, namely evolution.

Background

My past work shows that DNA origami can achieve custom-engineered synthetic cellular parts. However, the mere encapsulation of preformed parts conflicts with the vision of a self-replicating and evolving synthetic cell.

Proposed Methodology

I propose to produce and replicate functional RNA origami structures inside lipid vesicles (GUVs) by co-transcriptional folding from a DNA template.

Step 1: Genetic Encoding

First, I will genetically encode an RNA nanopore and RNA origami structure which induces GUV division. The DNA template (“genotype”) will determine the GUVs’ permeability and their division rate (“phenotype”).

Step 2: Directed Evolution

This genotype-phenotype mapping is the basis for directed evolution of the rationally engineered RNA origami structures. In particular, I will aim for efficient GUV division in repeated cycles of genetic diversification and selection.

Step 3: Continuous Evolution

In the third step, I will implement multiple growth and division cycles to enable continuous directed evolution. This will be achieved by system-level integration and laboratory automation of the directed evolution pipeline to iteratively reduce researcher intervention.

Expected Outcomes

Depending on externally applied selection pressures, continuous evolution will inevitably lead to the dominance of highly proliferating synthetic cells in mixed populations.

Significance

ENSYNC provides fundamental insights into evolutionary processes as well as applicable RNA origami-based tools for nanopore sensing and as genetically encoded biophysical probes in cell biology. Overall, ENSYNC pushes the boundaries of bottom-up synthetic biology to the point where synthetic cells can be evolved towards a distinct goal in biotechnology.